The overall goal of this project is to understand fundamental molecular mechanisms involved in membrane excitation phenomena such as visual transduction. The present project will be involved with the concurrent development of new methods for investigating membrane electrical phenomena and their application to the study of molecular events related to rhodopsin photoexcitation. The methods to be developed are based on spin labels, and new classes of hydrophobic ion spin labels will be prepared that will allow investigation of potentials and ionic currents in and across the membranes of small vesicles with high time resolution, as well as investigation of membrane capacitance and electrically active conformation transitions in membrane proteins. Applications to photoreceptors will be at both functional and structural levels. Functional studies will be directed toward a detailed description of the ionic conductances of the intact disc membranes and its possible regulation by rhodopsin, G protein (transducin) and cGMP. Of particular interest are Na+, K+ and Ca++. Studies of the ionic conductance of the plasma membrane will follow its isolation. Structural work will focus on the charge distribution in the rhodopsin molecule and its contribution to the electrostatic potential at the disc membrane surface. Both spin labeling and freeze-fracture methods will be employed. Finally, light and G protein induced conformational transitions in rhodopsin will be investigated by spin labeling methods. These studies will define conformationally active regions of the molecule.